This work was supported by NIH Grants R01-DC010146 and R01-DC010397. Its content is solely the responsibility of the authors and does not necessarily represent the official view of the National Institutes of Health.

1.细胞培养注意:所有的细胞培养协议必须使用适当的细胞培养技术来执行（参考见细胞生物学的第3章:实验室手册，第I卷10）。 HEI-OC1细胞不要求的适当附着和生长的细胞培养皿的任何附加涂层或处理。非常重要:不要用细胞培养的目的玻璃器皿菜肴;表型和细胞对药物的药物的生物反应会改变（G Kalinec＆F Kalinec，未发表）;建议使用传统的塑料细胞培养皿（见材料/设备表）。要特别注意无菌操作技术，以避免污染，但从来没有使用具有HEI-OC1细胞的抗生素（ 如氨苄青霉素或链霉素）。如果需要的话，可以使用的两性霉素B.边HeLa和HEK-293细胞已被用作在以前的研究与开平OC1 8，任何其他的细胞系能控制是为此目的可以接受的。 <ol><li>冷冻HEI-OC1细胞复苏 注意:此协议也可以与从本实验室开发的，如OC-K3，从科尔蒂11,12的器官的相同的转基因小鼠，和SV-K1其它细胞系使用的，从血管纹13,14。 <ol><li>从液态氮中取出冷冻开平OC1细胞的小瓶，并将其放置在水浴中在37℃。淹没只小瓶的下半部，并允许它解冻直到只有冰少量残留在小瓶中。擦拭用70％酒精小瓶的外部。 </li><li>来自小瓶吸取细胞和递送的全部体积（〜1.5×10 5个细胞/ ml）缓慢逐滴入含有10ml预温热的生长培养基中，如Dulbecco改进的Eagle培养基15毫升锥形管中（ DMEM）中，补充有10％胎牛血清（FBS）。 </li><li>通过离心管中，在300-500 XG除去DMSO 5分钟，丢弃吨他上清液并重新悬浮细胞于9ml新鲜生长培养基（DMEM + 10％FBS）的。由悬浮的细胞的磁通和回流分解团块或细胞的薄片，从吸管向培养基中，反之亦然，三至四次用吸管接触管的底部的前端。 </li><li>放置悬浮于生长培养基中毫米直径100未处理，塑料细胞培养皿的细胞的总体积。 </li><li>孵育所述细胞在33℃用10％的CO 2（在允许的条件）。 </li></ol></li><li> HEI-OC1细胞亚文化 注意:至汇合之前（〜80％）的细胞应使悬浮液和传代培养，以防止培养垂死。该协议也可以与来自相同的转基因小鼠在我们实验室开发的其它细胞系，如OC-K3，从科尔蒂11,12，和SV-K1的器官时，从血管纹13,14。 <ol><li>用2毫升的PBS除去旧培养基并洗涤细胞。&lt;/ LI&gt; <li>覆盖细胞单层用0.25％胰蛋白酶的溶液中，使用每表面积为25cm 2 1毫升到移动到下一个步骤的细胞的40％以上，必须分开。检查使用倒置显微镜的细胞，并且如果必要的，"拍击或轻按"培养皿轻轻释放任何残留的附着的细胞。 注:请注意胰酶消化作为长期可引起细胞死亡;没有足够和转移细胞就会不足计划的研究。 </li><li>悬浮细胞，按照1.1.3中描述的方法，并且将它们转移到15毫升锥形管中。 </li><li>离心机在300-500 XG 5分钟，弃去培养基，收集用新鲜的生长培养基中的细胞，并在种子他们，至少四个100毫米直径细胞培养皿。每盘的细胞数目将取决于回收的细胞的量。孵育所述细胞在33℃用10％的CO 2（P-HEI-OC1）并在必要时对分割再次多次计划的实验或用于生成新的股票。 </li><li>对于备料，取代250-550毫升的细胞培养瓶百毫米直径的菜肴;进行实验，较小的菜肴或多井板可能会更充足。 </li><li>对于差异化，孵化HEI-OC1细胞在允许的条件，直到他们达到〜80％汇合;然后移动的菜至39℃，5％的CO 2（NP-HEI-OC 1）为2至3周。 注:细胞将逐渐停止增殖和死亡。改变介质隔日以去除死细胞。根据原始数量的细胞，一般经过〜4周没有更多的细胞将可用于实验。当细胞被放置在NP条件下分化尽快启动，但不是每个单元格以同样的速度差异。重要的是，在分化过程prestin无表达和膜定位增加（见代表结果 ， 图4），提供质和分化水平的定量指示。 </li></ol></li></ol> 2.药物毒性研究注意:推荐用于这些研究（见代表结果 ， 图 1）在允许条件（P-HEI-OC 1）生长开平OC1细胞。 <ol><li>细胞活力（MTT法） <ol><li>收集的P-HEI-OC 1细胞通过以下在1.2中描述的方法，用任一自动细胞计数器或血细胞计数器计数。浓度调整到2.0×10 5个细胞/ ml。 </li><li>种子的细胞在96孔透明平底平板（每孔100μl），用于附连到衬底孵育过夜，然后与感兴趣的药物治疗。不要忘了，包括空格和非处理的细胞（对照）！ </li><li>药物治疗后（通常24或在33℃48小时），则执行根据制造商的协议MTT测定。一般来说，对于该测定的协议具有以下步骤: <ol><li>加入10微升的MTT试剂至各孔中。 </li><li>孵育在37℃的板2至4小时直到紫色染料是可见的，然后添加100微升的MTT洗涤剂试剂的每个孔中。不要摇晃板。 </li><li>覆盖板并使其保持在黑暗中进行2至4小时，在37℃。 </li><li>使用微量板读板器测量各孔在570nm的吸光度，包括空白（单独生长培养基）。利用在控制细胞存活率为100％的平均OD值正常化的数据。 </li></ol></li></ol></li><li>胱天蛋白酶3/7活化分析 注:建议这些研究在允许的条件（P-HEI-OC1）成长HEI-OC1细胞。 <ol><li>收集HEI-OC 1细胞通过以下在1.2中描述的方法，用任一自动细胞计数器或血细胞计数器计数。浓度调整到2.0×10 5个细胞/ ml。 </li><li>种子上WH细胞伊特-壁96孔板（每孔100μl），孵育过夜用于附着到衬底上，然后与感兴趣的药物治疗。不要忘了，包括空格和非处理的细胞（对照）！ </li><li>药物治疗后（通常24或在33℃48小时），则执行以下的各制造商的协议的胱天蛋白酶测定法。在一般情况下，对于该测定的协议具有以下步骤: <ol><li>准备试剂，它们搅拌均匀，并允许他们平衡至室温。 </li><li>除去从培养箱的细胞，并允许所述板平衡至室温。 </li><li>试剂的量表示每孔加入，小心通过不接触含有不同的样品具有相同的枪头井避免交叉污染。 </li><li>覆盖板，并用板振荡器在300-500转30秒混合。 </li><li>在室温下孵育该板的一段，至少30分钟。 DeterminË最佳的潜伏期经验。如果室内温度波动使用的恒温培养箱，因为温度波动会影响发光读数。 </li><li>测量每个发光良好，使用控制细胞平均发光的caspase激活的100％标准化值。 </li></ol></li></ol></li><li>细胞分裂（增殖） <ol><li>收集HEI-OC 1细胞通过以下在1.2中描述的方法，用任一自动细胞计数器或血细胞计数器计数。浓度调整到2.0×10 5个细胞/ ml。 </li><li>种子的细胞在96孔透明平底平板（每孔100μl）中，在用于附连到基片允许的条件下孵育过夜，然后与感兴趣的药物治疗。不要忘了，包括空格和非处理的细胞（对照）！ </li><li>处理后一小时，加至每孔1μl的准备100倍的BrdU（5-溴-2&#39;- deoxyuri用餐）溶液，并在33℃将板返回孵化器。 </li><li> 12，24和48小时后，取出从相应板中的现有的介质，并用PBS洗每个孔一次。 </li><li>执行按照制造商的协议的细胞增殖试验。在一般情况下，对于该测定的协议具有以下步骤: <ol><li>制备的试剂，其中包括定影/变性溶液，洗涤缓冲液，一级抗体检测溶液，二次抗体检测溶液，和BrdU溶液如制造商的方案表示。 </li><li>定影/变性溶液添加到各孔中，在制造商的方案中所示的量，并保持在室温下将板进行30分钟。 </li><li>拆下定影/变性溶液和在该制造商的协议所指示的浓度添加第一抗体。保持在室温下将板1小时。 </li><li>删除与主蚂蚁的解决方案ibody，洗涤平板用洗涤缓冲液3次，然后在该制造商的协议所指示的浓度添加二次抗体。保持与第二抗体的板在室温下30分钟。 </li><li>删除与第二抗体的溶液，冲洗板用洗涤缓冲液3次，并添加底物。 10分钟，在室温孵育后，添加一站式解决方案。请注意:控制颜色的变化;如果解决方案变得很暗，停止之前的10分钟的标准开发时的反应。 </li><li>在450 nm处加入终止液后的30分钟内读取吸光度。 </li></ol></li></ol></li><li>细胞毒性 注:建议这些研究在允许的条件（P-HEI-OC1）成长HEI-OC1细胞。 <ol><li>孵育在允许的条件，通常为24-48小时，开平OC1细胞单独细胞培养基（对照）或培养基加上感兴趣的药物。 </li><li>在治疗结束时，洗涤细胞三次，用PBS，并使用每3分钟一个非酶细胞解离溶液的表面积为25cm 21毫升分离。 </li><li>分离后，收集并在3000 xg离心离心沉淀细胞5分钟，除去上清液，并将细胞染色15分钟，在黑暗中与包括在细胞毒性测定试剂。 </li><li>通过流确定活开平OC1细胞的数目通过流式细胞仪的制造商仪所指示的。 </li></ol></li><li>衰老<ol><li>孵育在允许的条件，通常为24-48小时，开平OC1细胞单独细胞培养基（对照）或培养基加上感兴趣的药物。 </li><li>确定使用具有由已知提供可靠的结果Debacq-Chainiaux 等人 15或其它方法中描述的协议流式细胞仪的衰老相关的β-半乳糖苷酶阳性细胞的数目。流式细胞仪简要方案如下: <ol> &lt;li&gt;在的实验处理结束时，洗涤细胞三次，用PBS，然后用100nM巴弗洛霉素A1孵育它们在细胞培养基中在允许的条件1小时。 </li><li>添加C12FDG在〜33μM的终浓度，并继续培养细胞1-2小时。 </li><li>用PBS洗细胞两次，使用每3分钟一个非酶细胞解离溶液的表面积为25cm 21毫升收获它们，并在3000 xg离心离心沉淀他们5分钟。 </li><li>悬浮细胞在冰冷的PBS中，流式细胞仪通过流式细胞确定由流动的生产细胞仪指示正开平OC1细胞的数目。 </li></ol></li></ol></li><li>自噬<ol><li>收集HEI-OC1细胞控制和饥饿通过以下在1.2中描述的方法（剥夺血清的48小时），并对其进行处理用于印迹以下标准协议。在一般情况下，用于Western印迹的协议具有以下ST每股收益: <ol><li>种子HEI-OC 1细胞在6孔板以1.0×10 5细胞/ ml的浓度。 24和/或48小时后，洗细胞用冰冷的PBS中。 </li><li>裂解用200μlTNESV缓冲液（1％NP40，的50mM Tris -盐酸pH值7.5，100mM NaCl的，2mM的EDTA，1mM的钠3 VO 4）与蛋白酶抑制剂混合物和1mM PMSF在冰上5分钟的。 </li><li>收集细胞（由刮每个孔的底部），在4℃下转移到微量离心管并离心16,800×g离心5分钟。 </li><li>收集上清液并使用BCA测定法量化蛋白质浓度。 </li><li>添加5×上样缓冲液和1mM DTT向每个样品并煮沸5分钟以变性蛋白。 </li><li>运行使用4-12％凝胶上的SDS-PAGE的样品，并转移到PVDF膜上。 </li><li>用0.1％方框与在TBS-T的5％脱脂奶粉的膜吐温20在室温下60分钟。 </li><li>过夜孵育该膜在4℃下与伯蚂蚁ibodies。 </li><li>第二天，用TBS-T广泛洗涤该膜，并与缀合至辣根过氧化物酶（HRP）（1:1500）的次级IgG抗体孵育1小时。 </li><li>检测免疫反应性与增强的化学发光（ECL）检测系统。正常化使用GAPDH蛋白质表达水平（1:2,000在TBS​​-T中，在10％的脱脂奶粉）作为标准。 </li></ol></li><li>在Western印迹调查中，表达至少，自噬的两种不同的标记物，如自噬基因Beclin 1和LC3B（通常在1:1000稀释在TBS-T中，用2.5％BSA）中。不要忘记寻找如GAPDH或肌动蛋白表达这样的控制。 </li><li>评估通过使用数字印迹扫描器或计算机软件密度感兴趣的蛋白质的表达，如在公共领域NIH图像或的ImageJ（http://rsb.info.nih.gov/nih-image/）。 </li></ol></li></ol> 3.电生理学实验与HEI-OC1细胞<ol><li>收获细胞 注意:使用的细胞以50％-80％汇合生长100毫米直径的培养皿中。胰蛋白酶，的Accutase，不含酶的溶液，或b uffered 小号艾琳+ E thyleneðiamine 吨 ETRA 一个 cetic酸（PBS-EDTA） 芘 hosphate-可以用于提起细胞而不对gigaseals的数量和质量的影响显著。在某些情况下，但是，胰蛋白酶处理使得细胞更脆弱。在这些情况下，使细胞在开始实验前约30分钟恢复。 <ol><li>用10ml的PBS不含Ca 2+和Mg 2+洗细胞两次。 </li><li>在37℃下加入2毫升不含酶的分离器的溶液，如非酶细胞解离溶液中，并孵育菜3分钟，用5％ 的 CO 2。 </li><li>使用倒置显微镜来检查细胞的脱离。如果有必要，将CEL升培养盘分离更多的细胞，但做到这一点温和的。 注意:不要摇晃菜。 </li><li>加入10毫升的DMEM + 10％FBS和吸取细胞轻轻上下五次用10毫升移液管。 </li><li>看在显微镜下的细胞。如果细胞的&gt; 80％的已分离的（单），则不需要进一步的移液。如果细胞仍处于集群，重复轻轻吹打，直至超过80％的细胞是单身。 </li><li>所述〜10毫升悬浮细胞置于15毫升锥形管，离心机在100 xg离心2分钟，并弃去上清液。 </li><li>使用〜200微升的外部记录溶液（Leibovitz的L-15培养基调节至305 - 310毫渗透摩尔浓度用蒸馏水）以悬浮细胞。该细胞的光控应显示许多单身，圆形细胞具有光滑的膜边缘。 </li></ol></li><li>膜片钳和测量NLC <ol><li>执行膜片钳和使用电压依赖性的非线性电容的测量（NLC）dequate放大器和软件，以及标准电生理技术。内部（intrapipette）溶液使用150mM的氯化钾，1毫氯化镁 ，0.1毫EGTA，2毫摩尔ATP-Mg系，0.1毫GTP-钠，和10mM HEPES;调节pH值至7.2用Tris。 </li><li>在显微镜下，选择一个单一的，健康的细胞，与圆滑的边缘膜。附加玻璃吸管到细胞表面的前端，并检查膜电阻。这应该是周围3-6兆欧，对应于〜2微米的吸移管的尖端的内径（ID）。 </li><li>轻轻按压细胞质膜的枪头，然后应用吸。细胞膜的一部分将被吸入到移液管​​，产生在10顺序的电阻 - 100千兆欧姆（千兆欧密封）。 </li><li>通过破坏抽吸脉冲质膜建立全细胞的条件。 </li><li>使用不表达prestin无细胞如HEK-293，作为对照的9。 注:电流响应应该在5千赫进行过滤，并校正残余的串联电阻的作用的。可使用通常包含在锁相放大器的软件来执行的所有数据收集和多数分析。电容功能可以安装在与非线性电荷膜电压16的两状态波尔兹曼函数的一阶导数。 </li></ol></li></ol>

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The authors declare no existing or potential conflict of interest.

在本报告中，我们描述了如何培养开平OC1细胞，并利用它们来评价药物诱导的细胞毒性的机制，并调查prestin无，耳蜗外毛细胞的分子马达的功能特性。该技术程序，然而，有足够一般可以很容易地适应不同的研究。 这里描述的所有协议需要正确的使用成熟细胞培养技术10。就像任何其他细胞系，以开平OC1细胞工作，需要一个适当装备的细胞培养设备，具有认证生物安全柜，冷冻离心机，水浴，而对于细胞培养物检查1被反相显微镜计数细胞如果使用hemocytometric技术。一个独特要求，但是，是二加湿孵化器，一组在33°C / 10％CO 2的P-HEI-OC1细胞NP-HEI-OC1细胞的可用性和其他在39°C / 5％的CO 2秒。如果在计划的研究只涉及的P-HEI-OC1细胞，第二培养箱可以在37℃/ 5％CO 2的不同测定法来设置。 使用塑料细胞培养皿是用于与开平OC1细胞工作的一个重要的附加必要条件。事实上，开平OC1细胞是非常强大和它们将在不同的表面和非常不同的条件下生长。然而，它们的表型和对药物等刺激响应将强烈依赖于培养参数（G Kalinec＆F Kalinec，未发表）。正如我们在先前的纸8争辩，开平OC1细胞这可塑性可有利地用于设计新颖的实验。例如，众所周知，在耳蜗外淋巴中的氧张力比在海平面（〜21.3千帕）27的典型培养箱低得多的（〜2千帕）。在较低Ø孵化HEI-OC1细胞浓度2将改变他们的反应，也许使他们的审计更好的模型ORY感觉细胞。 应当强调的是，对于培养和HEI-OC1细胞传代培养所描述的协议也可以与来自相同的转基因小鼠在我们实验室开发的其他听觉细胞系，例如来自科尔蒂11,12和SV的器官的OC-K3被用于从-k1血管纹13,14。此外，这些细胞系可能为HEI-OC1细胞的实验中选择控制是有用的。例如，我们最近使用SV-K1细胞以证明成反比免疫反应prestin无和在开平OC1细胞的质膜的 Na + K + ATP酶抗体是细胞类型依赖性的响应，而不是与转基因小鼠从相关联这些细胞衍生的9。 与HEI-OC1细胞工作的其他关键的要求是适当的无菌技术，如清洁安全的机柜内所有的工作表面用70％E的做法thanol或异丙醇和用于程序所需的任何材料的去污。然而，虽然其它的哺乳动物细胞系是抵制不慎细菌污染，通过使用青霉素 - 链霉素或其他类似的组合经常保护，抗生素就不能同开平OC1电池中使用，除非该研究的目的是调查它们的影响。在无抗生素条件生成开平OC1细胞，并且它们必须避免，减少抗生素抗性细胞的出现。 最后，我们想再次强调一点已在本报告中多次提到:表型和HEI-OC1细胞对药物等刺激的反应将在很大程度上取决于文化的参数。因此，这是非常重要的是，与开平OC1细胞工作的实验室使用类似的协议和细胞培养条件，以便使它们的结果确实与其他基团提供的那些相当。此外，它是很重要的是要记住，HEI-OC1细胞系只是一个模型，与所有的模型具有的局限性。期待着与开平OC1细胞， 在体外实验将提供数据准确代表真实听觉的感觉细胞的体内反应是不现实的。但是，我们坚信，HEI-OC1细胞系用于研究听觉感官细胞的功能反应和药理学药物的潜在耳毒性的筛选有用的模型。

科尔蒂1的房子耳研究所-器官（HEI-OC1）细胞是从转基因小鼠1,2的听觉器官的。在33℃/ 10％CO 2（在允许的条件）从此转基因小鼠的任何细胞的温育诱导触发去分化和加速扩散的永生化基因的表达;移动单元至39℃/ 5％CO 2（非许可条件）导致减少的增殖，分化和，至少在开平OC1，细胞死亡2,3的情况。 HEI-OC1细胞克隆和表征在我们的实验室在十多年前，和最初的研究表明，它们表达耳蜗毛细胞，如prestin无，肌球蛋白7a中，Atoh1的，BDNF钙结合蛋白和钙调蛋白的特异性标志物，而且还支持标记细胞如连接蛋白26和成纤维细胞生长因子受体（FGF-R）2。因此，有人建议，HEI-OC1可以表示的commoñ祖为2尔蒂的器官感觉和支持细胞。平行研究提供了有力的证据表明，典型的耳毒性药物如顺铂，庆大霉素，链霉素诱导的caspase-3的活化在这些细胞中，而药物视为非耳毒性，如青霉素，没有2,3。因此，该细胞系被提出作为在体外系统来研究参与耳毒性及潜在耳毒性或新的药理学药物otoprotective性质筛选的细胞和分子机制。据估计，HEI-OC1细胞已经发表在过去十年超过150研究中使用。 而在寻找不同的药物的潜在促凋亡作用是最涉及该细胞系研究 ​​的主要目标，其他重要的细胞过程像自噬和衰老刚刚开始HEI-OC1细胞4-7进行调查。一世呐最近我们实验室8研究中，我们采用HEI-OC1细胞收集一套完整的有关细胞死亡，存活，增殖，衰老和自噬在临床上经常使用不同的药理学药物诱发的数据。我们还比较了一些开平OC1细胞与那些从HEK-293（人胚肾细胞）和HeLa（人上皮细胞）接受相同治疗的响应。我们的结果表明，开平OC1细胞的每种药物响应的特征的方式，具有鲜明的剂量和时间依赖的灵敏度所研究的机制中的至少一个。我们还强调，在这项研究的实验结果的正确解释，需要与多个技术进行8平行研究。 在不同的研究中，我们调查了采用HEI-OC1细胞来评价prestin无的功能反应，耳蜗外毛细胞马达蛋白（外毛细胞）9 </s了&gt;。我们报道流式细胞仪和激光扫描共聚焦显微镜研究上prestin无表达在容许（P-HEI-OC1）和非培养的HEI-OC1细胞的模式，以及非线性电容（NLC）和全细胞膜片钳研究容许（NP-HEI-OC 1）的条件。我们的结果表明，在NP-HEI-OC 1细胞以时间依赖性的方式既总prestin无表达和血浆膜定位增加。有趣的是，我们还发现，在含有的Na + K + ATP酶的减少，这从质膜易位到细胞质而不在总细胞表达显著变化相关的NP-HEI-OC1细胞的质膜的增加prestin无定位。此外，我们表明，P-HEI-OC 1细胞具有强大的NLC关联prestin无运动功能，当存在于质膜prestin无分子的密度增加，这降低。总之，这些结果强烈支持HEI-OC的用处1细胞研究听觉蛋白质。 在这个视频文章我们将描述如何培养开平OC1细胞，为什么它是方便使用的细胞在允许的条件（P-HEI-OC 1）细胞毒性的研究越来越大，如何评估药物诱导的细胞毒性，以及如何的机制/ s的执行电生理学研究（ 如 ，膜片钳，非线性电容（NLC））调查prestin无，耳蜗外毛细胞的分子马达的功能特性。

<table><tbody><tr><td>HEI-OC1 cells</td><td></td><td></td><td>ALL THE ASSAY KITS, EQUIPMENTS&amp;nbsp;</td></tr><tr><td>Class II Biological Safety cabinet</td><td>The Baker Company</td><td>Sterilgard III</td><td>AND COMPANIES INDICATED IN THE</td></tr><tr><td>Refrigerated centrifuge</td><td>Eppendorf</td><td>5810R</td><td>PREVIOUS 2 COLUMNS ARE ONLY</td></tr><tr><td>Inverted microscope</td><td>Zeiss</td><td>Axiovert 25</td><td>EXAMPLES, AND ANY OTHER SIMILAR</td></tr><tr><td>Waterbath</td><td>Stovall</td><td>HWB115</td><td>PRODUCT COULD BE USED.</td></tr><tr><td>Cell counter</td><td>Nexcelom</td><td>Cellometer Auto T4</td><td></td></tr><tr><td>Two (2) Cell incubators, one at 33 &amp;deg;C/10% CO&lt;sub&gt;2&lt;/sub&gt; and other at 39 &amp;deg;C/5% CO&lt;sub&gt;2&lt;/sub&gt;</td><td>Forma Scientific</td><td>3110</td><td></td></tr><tr><td>Cell culture dishes, PS, 100 mm x 20 mm with vents</td><td>Greinier Bio-One</td><td>664-160</td><td></td></tr><tr><td>Cell culture dishes , PS,&amp;nbsp; 60 mm x 15 mm with vents&amp;nbsp;</td><td>Greiner Bio-One</td><td>628160</td><td></td></tr><tr><td>Cellstar tissue culture flasks&amp;nbsp; 250 ml</td><td>Greiner Bio-One</td><td>658-175</td><td></td></tr><tr><td>Cellstar tissue cultur&amp;nbsp; flasks 550 ml</td><td>Greiner Bio-One</td><td>660-175</td><td></td></tr><tr><td>&amp;nbsp;6 well cell culture plate, with lid-Cellstar</td><td>Greiner Bio-One</td><td>657-160</td><td></td></tr><tr><td>Microtest Tissue culture plate, 96 well, flat bottom with lid</td><td>Becton Dickinson</td><td>353072</td><td></td></tr><tr><td>Micro-Assay-Plate, Chimmey, 96-well white, clear bottom</td><td>Greiner Bio-One</td><td>655098</td><td></td></tr><tr><td>50 ml Polypropylene conical tube with cap Cellstars</td><td>Becton Dickinson&amp;nbsp;</td><td>352070</td><td></td></tr><tr><td>15 ml Polypropylene conical tubes with cap-Cellstars</td><td>Greiner Bio-One</td><td>188-271</td><td></td></tr><tr><td>PBS pH 7.4 (1x)&amp;nbsp;</td><td>Life Technologies</td><td>10010-023</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium (DMEM)</td><td>Life Technologies</td><td>11965-084</td><td></td></tr><tr><td>Fetal bovine serum (FBS)&amp;nbsp;</td><td>Hyclone</td><td>SH10073.1</td><td></td></tr><tr><td>Leibovitz&#039;s L-15 Medium, no phenol red</td><td>Gibco/Invitrogen</td><td>21083-027</td><td></td></tr><tr><td>Trypsin, 0.25%&amp;nbsp;</td><td>Life Technologies</td><td>25200-056</td><td></td></tr><tr><td>TACS MTT Cell Proliferation Assay Kit</td><td>Trevigen</td><td>4890-25-K</td><td></td></tr><tr><td>Caspase-Glo 3/7 Assay&amp;nbsp; kit</td><td>Promega</td><td>&amp;nbsp; G8091&amp;nbsp;</td><td></td></tr><tr><td>BrdU Cell Proliferation Assay Kit&amp;nbsp;</td><td>Cell Signaling</td><td>6813</td><td></td></tr><tr><td>Non-enzymatic cell dissociation solution&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C5789</td><td></td></tr><tr><td>Cell-Tox Green Cytotoxicity Assay Kit</td><td>Promega</td><td>&amp;nbsp; G8741&amp;nbsp;</td><td></td></tr><tr><td>FACSAriaIII instrument&amp;nbsp;</td><td>BD Biosciences</td><td>&amp;nbsp;FACSAriaIII</td><td>With 488 nm excitation (blue laser)</td></tr><tr><td>Digital Blot Scanner</td><td>LI-COR</td><td>C-DiGit</td><td></td></tr><tr><td>Electrophoresis and Blotting Unit</td><td>Hoefer</td><td>SE300 miniVE</td><td></td></tr><tr><td>Spectra Max 5 Plate Reader with Soft Max Pro 5.2 Software</td><td>Molecular Devices</td><td>SpectraMax 5</td><td></td></tr><tr><td>Patch-clamp amplifier</td><td>HEKA</td><td>EPC-10</td><td></td></tr><tr><td>Puller for preparing patch electrodes</td><td>Sutter Instruments</td><td>P-97</td><td></td></tr></tbody></table>

working with auditory hei-oc1 cells

HEI-OC1 is one of the few mouse auditory cell lines available for research purposes. Originally proposed as an in vitro system for screening of ototoxic drugs, these cells have been used to investigate drug-activated apoptotic pathways, autophagy, senescence, mechanism of cell protection, inflammatory responses, cell differentiation, genetic and epigenetic effects of pharmacological drugs, effects of hypoxia, oxidative and endoplasmic reticulum stress, and expression of molecular channels and receptors. Among other several important markers of cochlear hair cells, HEI-OC1 cells endogenously express prestin, the paradigmatic motor protein of outer hair cells. Thus, they can be very useful to elucidate novel functional aspects of this important auditory protein. HEI-OC1 cells are very robust, and their culture usually does not present big complications. However, they require some special conditions such as avoiding the use of common anti-bacterial cocktails containing streptomycin or other antibiotics as well as incubation at 33 &#176;C to stimulate cell proliferation and incubation at 39 &#176;C to trigger cell differentiation. Here, we describe how to culture HEI-OC1 cells and how to use them in some typical assays, such as cell proliferation, viability, death, autophagy and senescence, as well as how to perform patch-clamp and non-linear capacitance measurements.

在一对夫妇最近的出版物中，我们报道了一整套旨在评估HEI-OC1细胞对几种常用药理学药物的反应，以及调查prestin无功能8,9研究。在这些研究中，我们利用了在前面部分中描述的所有协议。 一这些以前的研究的结果是，开平OC1细胞在非允许条件培养（39℃/ 10％的CO 2 = NP-HEI-OC 1）显示出在细胞活力高度显著下降，和一个同样显著增加在细胞死亡，对于在许可的条件下培养的细胞（33℃/ 10％的CO 2 = P-HEI-OC 1）（ 图1）。因此，任何毒性研究的NP-HEI-OC1细胞进行应致力于从文化条件的影响鉴别药物作用相当大的努力。在additioN，因为在细胞上的药物作用已垂死或具有降低存活率可以是不同的是对健康细胞的同种药物的效果，我们建议，使用P-HEI-OC 1细胞对于不特别要求分化的细胞的实验。 我们对不同药物的药理作用的研究也产生了有趣的结果。例如，顺铂，化疗药物经常用于治疗多种人类恶性肿瘤，和对乙酰氨基酚（APAP = N- 一 cetyl- PA RA-个p henol），销售最广过度的非处方止痛药在美国，分别为HEI-OC1细胞既有毒（ 图2）。然而，虽然APAP降低细胞活力和增加的胱天蛋白酶3/7活化，它不诱导细胞死亡。顺铂，与此相反，显著增加了三个响应（ 图2）。有趣的是，半胱氨酸蛋白酶3/7是mos晶体管逻辑Ÿ激活在较低的顺铂浓度，这表明低剂量顺铂诱导的细胞死亡可能是由介导的细胞凋亡，而高剂量的致命影响可以与胀亡/坏死的关联。而胀亡被定义为一个致命预途径，导致伴随着细胞和细胞器膨胀，起泡，以及增加的膜渗透性17,18不受调控的细胞死亡，坏死的是由启动相同的刺激活化的调节的非凋亡的细胞死亡机制细胞凋亡但类似的胀亡19-22的形态特征。 旨在调查的APAP，并在P-HEI-OC1细胞顺铂的效果额外研究表明APAP对细胞增殖的明确，剂量依赖性作用可以解释在细胞生存力没有在先前段落中所描述的细胞死亡的有趣的减少（ 图图3A）。顺铂阿尔斯<html> Ø降低细胞增殖，并与曝光即使小剂量（ 图3A）的时间其效果增强，但除了在纳入研究（ 图3B）所有的浓度和时间点降低衰老。在细胞衰老顺铂诱导的显著减少可能由细胞死亡的增加来解释，假设衰老细胞会是这样，也许，易发生死亡比健康细胞快23。 重要的是，这些代表性的结果表明，开平OC1细胞不同的药理学药物响应以鲜明的剂量和时间依赖的灵敏度所研究的机制中的至少一个。因此，任何实验结果的正确解释，需要调查的特定细胞过程有清晰的认识，并用来评估他们的技术每个人提供的信息。 jove_content"FO:保持-together.within页="1"&gt;共焦和流式细胞仪实验，另一方面，确认prestin无表达的P-HEI-OC1和NP-HEI-OC1细胞，用prestin无在immunolocalizing大多P-HEI-OC 1细胞（ 图4A中，箭头）的细胞质中，并且多集中在NP-HEI-OC 1细胞（ 图4B，箭头）质膜。流式细胞仪研究显示在NP-开平在prestin无表达明显增加-OC1细胞尊重到P-HEI-OC 1细胞（ 图 4C和4D）。在NP-HEI-OC1细胞prestin无表达和血浆膜定位的时间依赖性增加表明易位伴随更多prestin无分子的合成。然而，这些实验没有提供线索决定prestin无分子本来位于细胞质移动至质膜和新分子是否替换它们，如果它们保持在细胞质和仅新近合成esized prestin无移动到质膜，或者这两个过程的组合。 电生理学研究显示出强大的NLC中的P-HEI-OC 1细胞（ 图5B），具有峰值，该值降低，在高峰电容即转移到更高的去极化值的电压，当细胞被转移到非许可条件1和2周（ 图5C和5D）。因为prestin无从细胞质到质膜渐进易位，我们最初假设NLC将是NP-HEI-OC1比的P-HEI-OC1细胞更高，并且这将进一步在NP孵育时间而增加条件。令人惊奇的是，我们的研究结果表明完全相反的响应。我们推测，在质膜prestin无分子密度的增加可能会限制其运动功能。 欣页="1"&gt;注意在图5中在开平OC1细胞峰值电容电压的去极化尊重在外毛细胞，并在转染的HEK293细胞中24日报道的典型值;此外，观察到去极化偏移与时间NP条件增加。逐步去极化偏移类似于小鼠和大鼠发展25,26期间外毛细胞描述，并且它已被建议，它可能与其他细胞结构与OHC的质膜25，或一个成熟过程内在关联的发展到24 prestin无。 我们希望与HEI-OC1细胞研究较少的简短各显神通提供HEI-OC1细胞用于研究细胞和分子反应的广泛的潜在有用的足够的证据。 <html>广告/ 54425 / 54425fig1.jpg"/&gt; 图1: 细胞活性和细胞死亡的P-HEI-OC1和NP-HEI-OC1细胞 。 （A）细胞活力与MTT法进行评估。吸光度用酶标仪测量，并且在控制细胞平均OD值取作生存能力的100％。 （B）细胞死亡与细胞毒性检测评估。 530为15nm:阳性（死）细胞的数目通过流式细胞仪用488 nm激发（蓝色激光）和FL1确定。注意高度显著（P≤0.001）在细胞活力降低（A）和增加由非允许条件诱导的细胞死亡（B）。酒吧代表平均值（SEM）的标准误差。从参考8修改。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54425/54425fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="&lt;/ HTML">"图2"SRC ="/文件/ ftp_upload / 54425 / 54425fig2.jpg"/&gt; 图 2: 当由MTT法，胱天蛋白酶3/7和细胞毒性分析分析暴露于APAP及顺铂开平OC1细胞的响应 （A）的细胞存活率与MTT法评价，并吸光度用酶标仪测定。刽子手半胱氨酸蛋白酶3/7的（B）激活。 （C） 药物-用细胞毒性检测评估诱导的细胞死亡;正（死）细胞的数目通过流式细胞术确定的。在每一个实验的数据被归一化到相应的控制条件下（对照= 1），使可能发生的同步统计分析。 * = P≤0.05方面的控制。酒吧代表平均值（SEM）的标准误差。修改fromreference 8。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54425/54425fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> </一&gt; <img alt="图3" src="/files/ftp_upload/54425/54425fig3.jpg" /> 图3:HEI-OC1细胞"有丝分裂速度 ​​和衰老APAP顺铂的影响。 （A） 上HEI-OC1细胞有丝分裂，在24和​​48小时的速率药物引起的变化，用BrdU的测定评价。在开平OC1细胞（B）的药物诱导的衰老通过流式细胞术和senescence-测关联β半乳糖苷酶（SA-Bgal）技术。在每一个实验的数据被归一化到相应的控制条件下（对照= 1），使可能发生的同步统计分析。 * = P≤0.05方面的控制。酒吧代表平均值（SEM）的标准误差。修改fromreference 8。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54425/54425fig3large.jpg" target="_blank">请点击此处查看T的放大版本。</a>他的身影。 <img alt="图4" src="/files/ftp_upload/54425/54425fig4.jpg" /> 图4:在P-HEI-OC1和NP-HEI-OC1细胞prestin无表达-细胞与抗prestin无抗体标记和激光共聚焦显微镜和流式细胞仪观察 （ 一 ）P-HEI-OC1细胞prestin无免疫定位主要在细胞质（箭头）。 （B）在NP-HEI-OC1细胞，相反，prestin无反应浓缩在质膜（箭头）。 （C和D）流式细胞仪在2周时的NP条件（D）的培养开平OC1细胞的研究表明在prestin无表达相对于P-HEI-OC 1细胞（C）明显增加。 （C）插图是次要 AB只（阴性对照）。从参考9修改。请点击此处查看该图的放大版本。 <img alt="图5" src="/files/ftp_upload/54425/54425fig5.jpg" /> 图 5: 在P-HEI-OC1和NP-HEI-OC1细胞 （A）膜片钳HEI-OC1细胞NLC的研究 。 （B - D）NLC在P-HEI-OC1和NP-HEI-OC1细胞。注意NLC的峰的减少，并更去极化在NP-HEI-OC1细胞值曲线的渐进转变。从参考9修改。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54425/54425fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Working with Auditory HEI-OC1 Cells at JoVE.com

Working with Auditory HEI-OC1 Cells

House Ear Institute-Organ of Corti 1 (HEI-OC1) is one of the few mouse auditory cell lines currently available for research purposes. This protocol describes how to work with HEI-OC1 cells to investigate the cytotoxic effects of pharmacological drugs as well as functional properties of inner ear proteins.

与听觉HEI-OC1细胞工作

HEI-OC1 is one of the few mouse auditory cell lines available for research purposes. Originally proposed as an in vitro system for screening of ototoxic ...

Nanyang Technological University

Research

JoVE Journal

Biology

14.9K 次观看.  University of California, Los Angeles. House Ear Institute-Organ of Corti 1 (HEI-OC1) is one of the few mouse auditory cell lines currently available for research purposes. This protocol describes how to work with HEI-OC1 cells to investigate the cytotoxic effects of pharmacological drugs as well as functional properties of inner ear proteins.

视频: Working with Auditory HEI-OC1 Cells

Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells

Use of human induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC) for cell replacement therapies holds great promise. Several limitations including low yields and heterogeneous populations of differentiated cells hinder the progress of stem cell therapies. A fate restricted immortalized multipotent otic progenitor (iMOP) cell line was generated to facilitate efficient differentiation of large numbers of functional hair cells and spiral ganglion neurons (SGN) for inner ear cell replacement therapies. Starting from dissociated cultures of single iMOP cells, protocols that promote cell cycle exit and differentiation by basic fibroblast growth factor (bFGF) withdrawal were described. A significant decrease in proliferating cells after bFGF withdrawal was confirmed using an EdU cell proliferation assay. Concomitant with a decrease in proliferation, successful differentiation resulted in expression of molecular markers and morphological changes. Immunostaining of Cdkn1b (p27KIP) and Cdh1 (E-cadherin) in iMOP-derived otospheres was used as an indicator for differentiation into inner ear sensory epithelia while immunostaining of Cdkn1b and Tubb3 (neuronal &#946;-tubulin) was used to identify iMOP-derived neurons. Use of iMOP cells provides an important tool for understanding cell fate decisions made by inner ear neurosensory progenitors and will help develop protocols for generating large numbers of iPSC or ESC-derived hair cells and SGNs. These methods will accelerate efforts for generating otic cells for replacement therapies.

The current protocols to maintain immortalized multipotent otic progenitor (iMOP) cells and otic differentiation are described. Culture conditions and molecular markers that indicate differentiation into sensory epithelia and spiral ganglion neurons (SGN) are highlighted.

在永生多能耳科内皮祖细胞分化启动

Use of human induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC) for cell replacement therapies holds great promise. Several limitations ...

科研

发育生物学

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. The inner ear auditory sensory organ is critical for our ability to hear. It houses a highly specialized sensory epithelium that consists of mechano-transducing hair cells (HCs) and surrounding glial-like supporting cells (SCs). Despite structural differences in the auditory organs, molecular mechanisms regulating the development of the auditory organ are evolutionarily conserved between mammals and aves. In ovo electroporation is largely limited to early stages at E1 - E3. Due to the relative late development of the auditory organ at E5, manipulations of the auditory organ by in ovo electroporation past E3 are difficult due to the advanced development of the chicken embryo at later stages. The method presented here is a transient gene transfer method for targeting genes of interest at stage E4 - E4.5 in the developing chicken auditory sensory organ via in ovo micro-electroporation. This method is applicable for gain- and loss-of-functions with conventional plasmid DNA-based expression vectors and can be combined with in ovo cell proliferation assay by adding EdU (5-ethynyl-2&#180;-deoxyuridine) to the whole embryo at the time of electroporation. The use of green or red fluorescent protein (GFP or RFP) expression plasmids allows the experimenter to quickly determine whether the electroporation successfully targeted the auditory portion of the developing inner ear. In this method paper, representative examples of GFP electroporated specimens are illustrated; embryos were harvested 18 - 96 hr&#160;after electroporation and targeting of GFP to the pro-sensory area of the auditory organ was confirmed by RNA in situ hybridization. The method paper also provides an optimized protocol for the use of the thymidine analog EdU to analyze cell proliferation; an example of an EdU based cell proliferation assay that combines immuno-labeling and click EdU chemistry is provided.

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

The auditory organ mediates hearing. Here we present a modified in ovo micro-electroporation method optimized for studying auditory progenitor cell proliferation and differentiation in the developing chicken auditory organ.

基因转移到鸡听觉器官通过<em&gt;在卵</em&gt;微电

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. ...

Investigating Outer Hair Cell Motility with a Combination of External Alternating Electrical Field Stimulation and High-speed Image Analysis

OHCs are cylindrical sensorimotor cells located in the Organ of Corti, the auditory organ inside the mammalian inner ear. The name "hair cells" derives from their characteristic apical bundle of stereocilia, a critical element for detection and transduction of sound energy 1. OHCs are able to change shape &#x2014;elongate, shorten and bend&#x2014; in response to electrical, mechanical and chemical stimulation, a motor response considered crucial for cochlear amplification of acoustic signals 2.
OHC stimulation induces two different motile responses: i) electromotility, a.k.a fast motility, changes in length in the microsecond range derived from electrically-driven conformational changes in motor proteins densely packed in OHC plasma membrane, and ii) slow motility, shape changes in the millisecond to seconds range involving cytoskeletal reorganization 2, 3. OHC bending is associated with electromotility, and result either from an asymmetric distribution of motor proteins in the lateral plasma membrane, or asymmetric electrical stimulation of those motor proteins (e.g., with an electrical field perpendicular to the long axis of the cells) 4. Mechanical and chemical stimuli induce essentially slow motile responses, even though changes in the ionic conditions of the cells and/or their environment can also stimulate the plasma membrane-embedded motor proteins 5, 6. Since OHC motile responses are an essential component of the cochlear amplifier, the qualitative and quantitative analysis of these motile responses at acoustic frequencies (roughly from 20 Hz to 20 kHz in humans) is a very important matter in the field of hearing research 7.
The development of new imaging technology combining high-speed videocameras, LED-based illumination systems, and sophisticated image analysis software now provides the ability to perform reliable qualitative and quantitative studies of the motile response of isolated OHCs to an external alternating electrical field (EAEF) 8. This is a simple and non-invasive technique that circumvents most of the limitations of previous approaches 9-11. Moreover, the LED-based illumination system provides extreme brightness with insignificant thermal effects on the samples and, because of the use of video microscopy, optical resolution is at least 10-fold higher than with conventional light microscopy techniques 12. For instance, with the experimental setup described here, changes in cell length of about 20 nm can be routinely and reliably detected at frequencies of 10 kHz, and this resolution can be further improved at lower frequencies. 
We are confident that this experimental approach will help to extend our understanding of the cellular and molecular mechanisms underlying OHC motility.

A reliable method to investigate outer hair cell (OHC) motile responses, including electromotility, slow motility and bending, is described. OHC motility is elicited by stimulation with an external alternating electrical field, and the method takes advantage of high-speed image recording, LED-based illumination, and last generation image analysis software.

调查外毛细胞运动与外部的交流电场刺激和高速图像分析相结合

OHCs are cylindrical sensorimotor cells located in the Organ of Corti, the auditory organ inside the mammalian inner ear. The name "hair cells" derives ...

神经科学

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

听觉神经的光遗传学刺激

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as the hair cell. Basic research in the inner ear has led to a deep understanding of how the hair cell functions, and how dysregulation can lead to hearing loss and vertigo. For this research, the mouse has been the pre-eminent model system. However, mice, like all mammals, have lost the ability to replace hair cells. Thus, when trying to understand cellular therapies for restoring inner ear function, complementary studies in other vertebrate species could provide further insights. The auditory epithelium of birds, the basilar papilla (BP), is a sheet of epithelium composed of mechanosensory hair cells (HCs) intercalated by supporting cells (SCs). Although the anatomical architecture of the basilar papilla and the mammalian cochlea differ, the molecular mechanisms of inner ear development and hearing are similar. This makes the basilar papilla a useful system for not only comparative studies but also to understand regeneration. Here, we describe dissection and manipulation techniques for the chicken inner ear. The technique shows genetic and small molecule inhibition methods, which offer a potent tool for studying the molecular mechanisms of inner ear development. In this paper, we discuss in ovo electroporation techniques to genetically perturb the basilar papilla using CRIPSR-Cas9 deletions, followed by dissection of the basilar papilla. We also demonstrate the BP organ culture and optimal use of culture matrices, to observe the development of the epithelium and the hair cells.

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

The chick is a cost-effective, accessible, and widely available model organism for a variety of studies. Here, a series of protocols is detailed to understand the molecular mechanisms underlying avian inner ear development and regeneration.

在蛋 和 卵外 研究中研究鸟类内耳发育的方法

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as ...

Auditory Brainstem Response and Outer Hair Cell Whole-cell Patch Clamp Recording in Postnatal Rats

The outer hair cell is one of the two types of sensory hair cells in the mammalian cochlea. They alter their cell length with the receptor potential to amplify the weak vibration of low-level sound signal. The morphology and electrophysiological property of outer hair cells (OHCs) develop in early postnatal ages. The maturation of outer hair cell may contribute to the development of the auditory system. However, the process of OHCs development is not well studied. This is partly because of the difficulty to measure their function by an electrophysiological approach. With the purpose of developing a simple method to address the above issue, here we describe a step-by-step protocol to study the function of OHCs in acutely dissociated cochlea from postnatal rats. With this method, we can evaluate the cochlear response to pure tone stimuli and examine the expression level and function of the motor protein prestin in OHCs. This method can also be used to investigate the inner hair cells (IHCs).

This protocol describes methods to record the auditory brainstem response from postnatal rat pups. To examine the functional development of outer hair cells, the experimental procedure of whole-cell patch clamp recording in isolated outer hair cells is described step-by-step.

产后大鼠听觉脑干反应及外毛细胞全细胞贴片钳记录

The outer hair cell is one of the two types of sensory hair cells in the mammalian cochlea. They alter their cell length with the receptor potential to ...

小鼠耳蜗毛细胞的高效体外培养

Isolation and Culture of Primary Cochlear Hair Cells from Neonatal Mice

Cochlear hair cells are the sensory receptors of the auditory system. These cells are located in the organ of Corti, the sensory organ responsible for hearing, within the osseous labyrinth of the inner ear. Cochlear hair cells consist of two anatomically and functionally distinct types: outer and inner hair cells. Damage to either of them results in hearing loss. Notably, as inner hair cells cannot regenerate, and damage to them is permanent. Hence, in vitro cultivation of primary hair cells is indispensable for investigating the protective or regenerative effects of cochlear hair cells. This study aimed to discover a method for isolating and cultivating mouse hair cells. 
After manual removal of the cochlear lateral wall, the auditory epithelium was meticulously dissected from the cochlear modiolus under a microscope, incubated in a mixture consisting of 0.25% trypsin-EDTA for 10 min at 37 &#176;C, and gently suspended in culture medium using a 200 &#181;L pipette tip. The cell suspension was passed through a cell filter, the filtrate was centrifuged, and cells were cultured in 24-well plates. Hair cells were identified based on their capacity to express a mechanotransduction complex, myosin-VIIa, which is involved in motor tensions, and via selective labeling of F-actin using phalloidin. Cells reached &gt;90% confluence after 4 d in culture. This method can enhance our understanding of the biological characteristics of in vitro cultured hair cells and demonstrate the efficiency of cochlear hair cell cultures, establishing a solid methodological foundation for further auditory research.

作者聚焦：培养小鼠毛细胞用于听觉研究的进展 

Herein, we present a detailed protocol for isolating and culturing primary cochlear hair cells from mice. Initially, the organ of Corti was dissected from neonatal (aged 3-5 days) murine cochleae under a microscope. Subsequently, cells were enzymatically digested into a single-cell suspension and identified using immunofluorescence after several days in culture.

新生小鼠原代耳蜗毛细胞的分离和培养

Cochlear hair cells are the sensory receptors of the auditory system. These cells are located in the organ of Corti, the sensory organ responsible for ...

生物学

Preparing Wedge Brain Slices to Visualize Auditory Neurons

Source: Fischl, M. J. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/61664">In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity.</a>&#160;J. Vis. Exp.&#160;(2020)
This video demonstrates the process of preparing wedge-shaped brain slices in a laboratory setting. Using a vibratome, a wedge-shaped slice from a mouse&#39;s brain is crafted with varying thickness. This slice contains the auditory nerve root positioned on the thicker end, while the hearing-related neurons are located on the thinner end.

准备楔形脑切片以可视化听觉神经元

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Brain ...

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新生小鼠原代耳蜗毛细胞的分离和培养

准备楔形脑切片以可视化听觉神经元

与听觉HEI-OC1细胞工作

摘要

探索更多视频

此视频中的章节

在永生多能耳科内皮祖细胞分化启动

基因转移到鸡听觉器官通过

调查外毛细胞运动与外部的交流电场刺激和高速图像分析相结合

听觉神经的光遗传学刺激

在蛋 和 卵外 研究中研究鸟类内耳发育的方法

产后大鼠听觉脑干反应及外毛细胞全细胞贴片钳记录

新生小鼠原代耳蜗毛细胞的分离和培养

新生小鼠原代耳蜗毛细胞的分离和培养

新生小鼠原代耳蜗毛细胞的分离和培养

准备楔形脑切片以可视化听觉神经元

在蛋和卵外研究中研究鸟类内耳发育的方法